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1 Department of Pharmacology, Nippon Medical School, Tokyo 113-8602, Japan
2 Waseda-Olympus Bioscience Research Institute, Helios, no. 05-01/02, 11 Biopolis Way, Singapore 138667
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(Received 2 February 2005;
accepted after revision 22 March 2005;
first published online 24 March 2005)
Corresponding author S. Konishi: Waseda-Olympus Bioscience Research Institute, Helios, no. 05-01/02, 11 Biopolis Way, Singapore 138667. Email: skonishi{at}waseda.jp
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Introduction |
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In the cerebellum, it was previously reported that monoamines, such as noradrenaline (NA) and serotonin, enhance GABAergic transmission between the interneurone basket cells (BCs) and Purkinje cells (PCs) (Llano & Gershenfeld, 1993; Mitoma et al. 1994; Mitoma & Konishi, 1996; Kondo & Marty, 1997). Subsequently, the NA-induced modulation at cerebellar GABAergic synapses has been ascribed to activation of ß2-adrecoceptors in presynaptic BC nerve terminals (Mitoma & Konishi, 1999; Saitow et al. 2000), and the actions of exogenously applied NA could be mimicked by synaptically released monoamines following stimulation of afferent fibres in the cerebellar cortex (Mitoma & Konishi, 1999). Furthermore, our recent study has shown that noradrenergic enhancement of cerebellar GABAergic synapses was mediated by two different actions (Saitow et al. 2000): (1) cAMP formation following ß2-adrecoceptor activation excited BCs through acceleration of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel, resulting in an increase in the frequency of spontaneous inhibitory postsynaptic currents (IPSCs); and (2) there was, additionally, an HCN channel activation-independent increase in the amplitude of evoked IPSCs. It has been demonstrated also that depending on the increase in the amount of NA released into the BC–PC synapse, the presynaptic ß-adrenoceptor-mediated modulation of GABA release may cause a shift from short-term to long-term potentiation (LTP) (Mitoma & Konishi, 1999; Saitow et al. 2000).
In this study, we investigated the mechanisms underlying the noradrenergic LTP of GABAergic transmission, including the potential involvement of HCN channels (Beaumont & Zucker, 2000; Mellor et al. 2002). Our findings suggest that noradrenergic and ß-adrenoceptor-mediated LTP of GABA release at cerebellar inhibitory synapses involves a cAMP–protein kinase A (PKA)-dependent signalling pathway but does not depend on cAMP-regulated HCN channel modulation. Furthermore, our data provide evidence that PKA-dependent enhancement of Ca2+ sensor function in the GABA release machinery and increase in the number of readily releasable synaptic vesicles contribute to this form of long-term synaptic plasticity of GABAergic transmission in the cerebellar cortex. Part of the present study has appeared in abstract form (Saitow & Konishi, 2002).
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Methods |
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Experiments were performed using thin slices of the cerebellum prepared from 12- to 18-day-old Wistar rats (Saitow et al. 2000) with a protocol approved by the Ethics Review of Nippon Medical School. Animals of either sex were deeply anaesthetized with halothane inhalation (approximately 2% v/v) and their brains were rapidly removed. Parasagittal slices (250 µm thick) were cut by using a vibratome (VT1000S, Leica, Germany) at 
4°C in a Na+-deficient saline that contained (mM): sucrose 299.2, KCl 3.4, CaCl2 0.3, MgCl2 3.0, Hepes 10, NaH2PO4 0.6 and glucose 10. This solution appeared to help avoid tissue damages due to excessive excitation during slicing. The slices were kept for more than 1 h in a humidified and oxygenated chamber with an interface of artificial cerebrospinal fluid (ACSF) that contained (mM): NaCl 138.6, KCl 3.4, CaCl2 2.5, MaCl2 1.0, NaHCO3 21.0, NaH2PO4 0.6 and glucose 10.0. The pH of the ACSF was maintained at 7.4 by bubbling with 95% O2–5% CO2. In most experiments, slices were superfused with ACSF in which 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was added at 10 µM to eliminate glutamatergic excitatory synaptic responses.
Individual slices were transferred to a recording chamber attached on the stage of a microscope (BX50WI, Olympus, Japan) and continuously perfused with the oxygenated ACSF at a flow rate of 1.5 ml min–1 and temperature of 25–27°C. Patch electrodes used for whole-cell voltage-clamp recordings from PCs had resistances of 2–4 M
when filled with an internal solution that contained (mM): CsCl 140.0, KCl 5.0, K-EGTA 0.1, Na-Hepes 5.0, Mg-ATP 3.0, Na-GTP 0.4 (pH 7.4). PCs were visually identified under Nomarski optics with a water-immersion objective (63 x, NA, 0.90, Olympus, Japan). The membrane potential recordings from BCs were made in the tight-seal whole-cell patch clamp. Glass electrodes used for this purpose had resistance of 5–7 M when filled with an internal solution that contained (mM): potassium methanesulphonate 150.0, KCl 5.0, K-EGTA 0.1, Na-Hepes 5.0, Mg-ATP 3.0, Na-GTP 0.4 (pH 7.4). Membrane currents and potentials were recorded with a patch-clamp amplifier (EPC-9, HEKA Elektronik, Darmastadt, Germany). The series resistance monitored through experiments was in the range from 10 to 20 M and was not compensated. Data were discarded if this value changed by more than 30%. Data were analysed using the software PULSEFIT (HEKA Elektronik) and KYPOLT (http://www.kyence.com). Miniature synaptic currents were visually inspected, and peak amplitudes were determined for analysis using the software Mini analysis (Synaptosoft, Decatur, GA, USA). Data were also continuously stored during experiments on a videotape recorder after digitizing by a pulse code modulation data recorder (NF Electronic Instruments, Japan). All signals were filtered at 1 kHz and sampled at 2 kHz. Membrane currents were recorded from PCs at a holding potential of –50 mV. IPSCs were evoked every 15 or 30 s by stimulation (10–30 V, 60–100 µs) via glass microelectrodes (tip diameter, 1–2 µm) filled with ACSF and placed within the molecular layer. Evoked IPSCs were abolished by bicuculline (10 µM), indicating that they are mediated by GABAA receptor activation.
To examine the properties of the readily releasable pool (RRP) in BC terminals, a hypertonic solution containing 500 mM sucrose in ACSF was puff-applied to the vicinity of recorded PCs through a micropipette controlled by a picospritzer (PV830, WPI, Sarasota, Fl, USA). In these experiments, slices were superfused with an ACSF containing 10 µM CNQX, 50 µM D-2-amino-5-phosphonopentanoic acid (D-APV) and 1 µM CGP55845to block glutamatergic excitatory and GABAB receptor-mediated inhibitory synaptic responses. In the experiments where the effects of PKA inhibitors were examined, slices were preincubated for at least 1 h in a submerged-type chamber with an ACSF that contained either 5 µM H-89 or 100 µM Rp-Adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS). After transfer to the recording chamber, the slices were continuously superfused with the H-89- or Rp-cAMPS-containing ACSF.
Chemicals used in this study were obtained from the following sources: bicuculline, isoprenaline (isoproterenol; ISP), Rp-cAMPS and H-89 from Sigma (St Louis, MO, USA); CNQX, ZD7288, D-APV and CGP55845from Tocris Cookson (Bristol, UK); tetrodotoxin (TTX) from Sankyo (Tokyo, Japan); and picrotoxin from Wako (Tokyo, Japan).
Measurements of presynaptic Ca2+ influx
Digital fluorescence imaging was carried out using the water-immersion objective (63 x, NA, 0.90) on a Fluoview confocal microscope system (Olympus, Tokyo, Japan) mounted on the microscope. BCs were loaded with either 400 µM Calcium Green-5N, a low-affinity Ca2+ indicator, or 200 µM Oregon Green 488 BAPTA-1, a high-affinity indicator (Molecular Probes, Eugene, OR, USA) added in the potassium-based patch-pipette solution (see above). Approximately 15–20 min after indicator loading into BCs with the whole-cell mode, local areas of contacts between stained BC axons and PC somata were identified by visual inspection during image acquisition. Fluorescence was excited using the 488-nm line of an argon laser, and emissions were detected through a 515-nm low-pass filter. Fluorescence time courses were then recorded in line-scan mode by positioning the scan line across the identified synaptic contacts. Fluorescence signals were sampled at rate of 2.5 ms per line. Image acquisition was started while the cell was held at –60 mV and a train of three pulses (each 3 ms to 0 mV) was given at 150 ms after the onset of imaging. This protocol was repeated 5–10 times every 1 min before and during application of ISP. Recorded signals were averaged to reduce the signal-to-noise ratio. [Ca2+]i changes are expressed as the ratio of the change in fluorescence over the resting fluorescence level (
F/F0). Under these conditions, there was no significant attenuation of resting fluorescence level (F0) during the recording if BCs were not stimulated by depolarization pulses. Data were discarded when F0 was too low to detect even with detectable signal increases following depolarization-pulse stimulation (see Fig. 3A).
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Numerical data are given as means ± S.E.M., where n represents the number of independent experiments. Statistical differences were evaluated using Student's paired t test unless otherwise noted. For multiple comparisons between experimental groups and comparison of cumulative curves, Tukey's parametric multiple comparison test and Kolmogorov-Smirnov (K-S) test were used, respectively.
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Results |
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As previously reported, stimulation of BCs with the ß-adrenoceptor agonist ISP elicited two actions on the GABAergic transmission that were recorded in PCs: a short-term increase in the frequency of spontaneous IPSCs (sIPSCs) and LTP of stimulation-evoked IPSCs (eIPSCs) (Mitoma & Konishi, 1999; Saitow et al. 2000). The ISP-induced LTP of GABAergic IPSCs was illustrated in Fig. 1A: the amplitude of eIPSCs increased to 131.2 ± 7.5% of baseline following application of ISP (20 µM, n = 19, determined at 5–10 min after starting the ISP application), and the enhancement persisted for at least 40 min after washing out the ß-agonist. The ISP-induced LTP was unlikely to have resulted from persistent activation of ß-adrenoceptors by the ß-agonist, because application of the ß-adrenoceptor antagonist propranolol (10 µM) did not attenuate the long-lasting effect of ISP on eIPSCs (• in Fig. 1A). There was no significant difference in changes of the eIPSC amplitude between ISP alone and ISP plus propranolol determined at 35–40 min after the ISP application (P > 0.5, unpaired t test, n = 7).
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ZD7288 itself reduced the eIPSC to 76.5 ± 1.1% when determined at 15–25 min after ZD7288 application (•, Fig. 1D; n = 7). This effect was attributable to its presynaptic action on the BCs, because the inhibitor significantly increased the paired-pulse ratio (PPR) of eIPSCs from 0.96 ± 0.05 to 1.36 ± 0.11 (n = 7, P < 0.01; see traces labelled a and b in Fig. 1D). Furthermore, the HCN channel inhibitor hyperpolarized BCs (10.7 ± 1.5 mV, n = 4), resulting in inhibition of repetitive firings (Fig. 1B). On the other hand, there were no significant changes in the extent of ISP-induced enhancement in the presence or absence of ZD7288 (Fig. 1D and E); the percentage increases in eIPSC amplitude were 149 ± 10% in the control solution (n = 12) and 154 ± 12% in the presence of ZD7288 (n = 7, determined at 5–10 min after starting the ISP application; P > 0.8, unpaired t test). Although the amplitude of eIPSCs slightly decreased following ISP washout and again increased after washing out ZD7288 (Fig. 1E), it is not clear whether the HCN inhibitor has a reversible suppressive action on GABAergic synapses. Together, these findings showed that HCN channel activation in BCs contributed only to the ISP-induced increase in the sIPSC frequency but not to the enhancement of stimulation-evoked GABA release at BC–PC inhibitory synapses.
As ß-adrenoceptors are known to be linked with cAMP–PKA-dependent mechanisms, we then examined the effects of the PKA inhibitor, H-89, and the membrane permeable cAMP analogue, Rp-cAMPS, on the ISP-induced LTP of GABAergic transmission at the BC–PC synapse. Pretreatment with either H-89 (5 µM) or Rp-cAMPS (100 µM) almost completely abolished the ISP-induced long-term enhancement of eIPSCs in PCs (142.8 ± 2.1 (Fig. 2A, n = 6) and 140.8 ± 1.9% of baselines (Fig. 2B, n = 19) in the control solution, and 96.6 ± 1.8 (n = 8, P < 0.01) or 97.0 ± 1.7% of baselines (n = 12, P < 0.001, unpaired t test, assessed at 5–10 min after starting the ISP application) in the presence H-89 or Rp-cAMPS, respectively). The results point to the involvement of the cAMP–PKA cascade in this form of ß-adrenoceptor-mediated LTP at inhibitory synapses. In contrast, H-89 at the same concentration did not affect the ISP-induced increase in HCN channel activation (Fig. 2C, 125.6 ± 16% of control, n = 9), suggesting that HCN channel modulation by ß-adrenoceptors depended on a direct action of cAMP on HCN channels. This interpretation was supported by the results presented in Fig. 2D. Rp-cAMPS (50 µM), a cAMP analogue and PKA inhibitor, mimicked the action of ISP in increasing the amplitude of HCN currents in BCs: the HCN current amplitudes were 105.7 ± 2.0 pA in the control condition (n = 14) and 168.8 ± 1.8 pA after Rp-cAMP injection (n = 18, P < 0.001 assessed during the period 11–20 min after the initiation of recordings). Taken together, these findings indicated that the ß-adrenoceptor activation at BC–PC inhibitory synapses elicited two distinct actions on GABAergic transmission: (1) the increase in eIPSC amplitude that depended on a cAMP–PKA cascade; and (2) the increase in sIPSC frequency that resulted from a direct action of cAMP on HCN channels to accelerate firing rate due to the excitability increase in presynaptic BCs (see also Saitow & Konishi, 2000).
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ß-Adrenoceptor activation increases the activity of voltage-dependent Ca2+ channels in various excitable cells (Muraki et al. 1993; Gao et al. 1997) and enhances excitatory synaptic transmission by increasing the open probability of P/Q-type Ca2+ channels in presynaptic terminals (Huang et al. 1998). Therefore, we examined whether the ß-agonist ISP could enhance Ca2+ influx into BC axon terminals by measuring the action potential-triggered Ca2+ transients in individual boutons of BC axons loaded with Ca2+-sensitive fluorescence indicators and measuring [Ca2+]i with fast confocal imaging in a line scan mode. A representative image of action potential-induced Ca2+ transients, measured by using a high-affinity Ca2+-indicator, Oregon Green BAPTA-1 (OGB-1), is illustrated in Fig. 3A. In agreement with a previous report (Llano et al. 1997), PC somata were surrounded by fluorescent boutons along the trajectory of the labelled BC axon. Depolarization of BC soma (from –60 mV to 0 mV for 3 ms) caused escaping currents (Fig. 3B) that induced action potentials, which in turn triggered reproducible Ca2+ transients in these boutons (Fig. 3A). The peak amplitude of depolarization-induced Ca2+ transients (F/F0) augmented with increasing either extracellular Ca2+ concentrations from 2.5 mM to 5 mM (Fig. 3C) or the number of depolarizing pulses to stimulate BCs (Fig. 3D). However, when [Ca2+]i was determined by the use of either OGB-1 (data not shown) or a low-affinity indicator, Calcium Green 5N (CG5N), the ß-agonist ISP did not alter Ca2+ transients at individual boutons (Fig. 3E): the peak amplitudes were 19.3 ± 3.4 and 18.7 ± 4.2% in the absence and presence of ISP, respectively, n = 8, P > 0.7. Furthermore, there was no significant change in the decay time constants of Ca2+ transients before and after the application of ISP (92.5 ± 28 and 102.3 ± 20 ms in the absence and presence of ISP, respectively; n = 8, P > 0.6). The resting level of fluorescence was not significantly changed by ISP (n = 4, data not shown). These results indicated that the long-term modulation of GABA release following ß-adrenoceptor activation at BC–PC synapses was independent of calcium influx and, perhaps, of changes in [Ca2+]i dynamics, such as buffering and removal of Ca2+ ions in BC axon terminals.
Effects of ß-adrenoceptor activation on the GABA release machinery
Based on the above results, we then examined whether the ß-adrenoceptor activation could modulate the exocytotic machinery in GABAergic presynaptic terminals. It has been previously reported that serotonin increases the frequency of miniature excitatory synaptic responses in the crayfish neuromuscular junction through activation of HCN channels in the presynaptic terminals (Beaumont & Zucker, 2000). However in the cerebellar BC–PC inhibitory synapses, ISP increased the frequency of mIPSCs even in the presence of the HCN channel inhibitor ZD7288 (Fig. 4A), and this effect was statistically significant (Fig. 4B, P < 0.05 K-S test and Fig. 4D, P < 0.001, n = 14). ISP did not significantly affect the mean amplitude of mIPSCs (Fig. 4C, P > 0.5 K-S test and Fig. 4E, P > 0.3, n = 14). These observations suggested that ß-adrenoceptor activation directly modulates the release machinery in presynaptic BC nerve terminals.
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Because neurotransmitter release is triggered by the action potential-induced rise of intracellular Ca2+ concentrations in presynaptic terminals (Augustine et al. 1999), we addressed how the dependency of GABAergic transmission on Ca2+ concentration could be modulated by the ß-adrenoceptor activation. Thus we examined the relationship between the average amplitudes of eIPSCs in PCs and changes in [Ca2+]o before and after application of ISP. Fitting the Hill equation to this relationship in the absence of ISP showed that the change of eIPSC amplitude depended on the second power of [Ca2+]o (Fig. 6A). To date the efficacy of excitatory transmission has been shown to depend on the fourth power of [Ca2+]o at the neuromuscular junction (Dodge & Rahamimoff, 1967) and central excitatory synapses (Yawo, 1996), whereas GABAergic transmission at collicular inhibitory synapses has been reported to display an approximately third-power dependency on presynaptic Ca2+ concentration (Kirischuk et al. 1999). As illustrated in Fig. 6A, the extent of ß-adrenergic agonist-induced enhancement of eIPSCs was consistently larger at lower [Ca2+]o (210 ± 12% and 123 ± 3% at 1 and 5 mM [Ca2+]o, respectively, n = 8–9). Consequently, ISP shifted the relationship between the eIPSC amplitude and [Ca2+]o in a leftward direction with a decrease in Kd for Ca2+ from 1.9 to 1.0 mM (Fig. 6A), suggesting that the increase in Ca2+ sensitivity of the release machinery contributed to the ß-adrenoceptor-mediated enhancement of inhibitory transmission at BC–PC synapses. Furthermore, the ISP-induced enhancement of the Ca2+ sensor function depended on a cAMP–PKA cascade, because the PKA inhibitor Rp-cAMPS (100 µM) markedly suppressed the ISP-induced increase in the eIPSC amplitude at 1.5 and 2.5 mM [Ca2+]o (Fig. 6B): 210.0 ± 12.0% (n = 10) and 144.3 ± 13.0% (n = 5) increases in the absence and presence of Rp-cAMPS at 1.5 mM [Ca2+]o, respectively (P < 0.01, unpaired t test).
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We next examined whether ß-adrenergic agonist stimulation would affect the process of neurotransmitter recruitment that regulates the size of RRP (namely, the number of releasable vesicles) in GABAergic terminals. In the experiments illustrated in Fig. 7, the time course of changes in GABAergic transmission following repeated activation of the presynaptic terminals was compared before and after ISP application. Tetanic stimulation at 20 Hz for 1.5 s (30 stimuli) in the control solution transiently reduced the amplitude of eIPSCs within the initial 10 stimuli, reaching a steady level of its amplitude with only partial suppression. This effect was prominent in 5 mM [Ca2+]o (Fig. 7A and B). Possible explanations for such changes in GABA release kinetics following tetanic stimulation are that: (1) the presynaptic terminals of cerebellar interneurones might be endowed with relatively large size of the RRP, which could be replenished during repeated activation; and/or (2) the Ca2+ sensor in the release machinery of BC terminals could support sustained GABA exocytosis. Under these conditions, application of ISP not only reinforced GABA release induced by individual stimuli but also enhanced the extent, as well as the speed, of transmission depression during repeated stimulation at 1.5 and 2.5 mM [Ca2+]o, but the effects were less prominent at 5 mM [Ca2+]o (Fig. 7A and B).
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In addition to increasing the RRP size, ISP also increased the rate at which the transmitter pool was refilled following the high frequency stimulation (Fig. 7E and F). To determine the time course of refilling, we measured the amplitude of IPSCs evoked by single test stimuli at different intervals after termination of high-frequency train stimulation (20 Hz with 30 stimuli). While the overall time course of IPSC recovery in the control experiment was fitted by a single-exponential function with a time constant of 210.4 ± 21.4 ms (n = 10), the ISP application significantly accelerated the refilling kinetics with a time constant of 86 ± 11.6 ms (P < 0.05, unpaired t test, n = 9). These observations imply that (1) ß-adrenoceptor activation could principally modulate the Ca2+ sensitivity of the release machinery, contributing to the increase in eIPSC amplitude and (2) that the ISP-induced increase in the RRP would serve as a secondary mechanism for compensating vesicle depletion following the increment of release probability. Based on these results, we suggest that the ß-adrenoceptor stimulation at BC–PC synapses enhanced GABA release through the up-regulation of both the Ca2+ sensor in the exocytotic machinery and the size of transmitter pools immediately available for release.
ß-Adrenoceptor stimulation enhances synchronous and asynchronous GABA release
Finally, we explored the effects of ß-adrenoceptor activation on GABA release kinetics. The process of neurotransmitter release is divided into phasic synchronous and tonic asynchronous phases. The tonic release is manifest when Ca2+ ions are replaced by Sr2+ ions at both excitatory (Atluri & Regehr, 1996) and inhibitory synapses (Rumpel & Behrends, 1999). A previous study on the knockout mice lacking synaptotagmin I, a presynaptic Ca2+-binding protein, has demonstrated that this Ca2+-binding protein contributes to synchronous release of glutamate at hippocampal excitatory synapses without affecting asynchronous release (Geppert et al. 1994). It is therefore possible that the two release mechanisms could be differentially modulated by the ß-adrenoceptor activation. This possibility was tested in the experiments shown in Fig. 8. When Ca2+ in the ACSF was replaced by an equimolar amount of Sr2+, the amplitude of eIPSCs (generated by synchronous GABA release) was largely suppressed (Fig. 8A and B, 31 ± 4% of the control; P < 0.001, Tukey's multiple comparison test, n = 11), whereas asynchronous GABA release became prominent (Fig. 8D and F, 0.4 ± 0.7 and 9.6 ± 1.2 Hz in Ca2+- and Sr2+-containing ACSF, respectively, P < 0.001 Tukey's multiple comparison test, n = 11). ISP (20 µM) not only increased the synchronous component of GABA release in the Sr2+-containing ACSF (Fig. 8A–C, 153.8 ± 8.6% of baseline determined at 3 min after ISP application; P < 0.05, Tukey's multiple comparison test, n = 11) but also enhanced asynchronous GABA release as determined by comparing inhibitory synaptic events during a time window of 500 ms before (Pre) and after (Post) single shock stimulation (Fig. 8D–F, 9.1 ± 1.1 and 13.0 ± 1.3 Hz before and after ISP application in Sr2+-containing ACSF, respectively; P < 0.05, Tukey's multiple comparison test, n = 10). These results indicated that both Ca2+- and Sr2+-mediated release mechanisms, namely synchronous and asynchronous GABA release at BC–PC inhibitory synapses, were under the control of ß-adrenoceptor-mediated modulation.
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Discussion |
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For cerebellar GABAergic interneurones, the short-term modulation of their excitability by ß-adrenoceptor stimulation depended only on the cAMP-mediated HCN channel acceleration but not on the cAMP–PKA pathway (Saitow & Konishi, 2000), whereas, as the present results suggested, increase in eIPSC amplitude (i.e. LTP of GABA release), largely relied on a cAMP–PKA cascade. Stimulation of afferent fibres in the cerebellar cortex mimicked the ß-adrenoceptor-mediated LTP of GABAergic transmission at BC–PC synapses (Mitoma & Konishi, 1999). Therefore, depending on different time windows and signalling pathways following monoaminergic afferent activation, the ß-adrenergic short- and long-lasting forms of plasticity in the GABAergic inhibitory synapse provides a likely mechanism of feedforward inhibition onto PCs, the sole output from the cerebellar cortex, thereby critically influencing motor coordination in the cerebellar function.
Our findings revealed two independent mechanisms underlying the noradrenergic and ß-adrenoceptor-mediated LTP of inhibitory GABAergic transmission at BC–PC synapses: ß-adrenoceptor stimulation not only up-regulated the efficacy of the Ca2+-sensor in the GABA release machinery, it also increased the number of presynaptic vesicles that were immediately available for release.
ß-Adrenoceptor activation regulates the Ca2+ sensor function and the number of releasable synaptic vesicles
Using two independent tests, we have shown that the ß-adrenoceptor stimulation enhanced the GABA release machinery in the presynaptic nerve terminals: (1) the ß-agonist ISP increased the frequency of mIPSCs in PCs without significantly changing the mean amplitude, and this effect persisted in the presence of the HCN channel inhibitor ZD7288 (Fig. 4); and (2) ISP also increased GABA release produced in response to hypertonic stimulation of the nerve terminal in a manner sensitive to PKA-linked pathways but insensitive to HCN channels (Fig. 5).
Our data from two other experiments contributed to the characterization of the targets involved in the noradrenergic LTP of GABA release at BC–PC synapses. First, the dependency of GABAergic transmission on [Ca2+]o was reduced by ISP (Fig. 6), indicating that ß-adrenoceptor stimulation enhanced the efficacy of the Ca2+ sensor in the GABA release machinery. The Hill equation fitted to the relationship between the eIPSC amplitudes and [Ca2+]o indicated that the only parameter affected by ISP application was Kd, the dissociation constant of the release machinery to Ca2+ ions, which was reduced by ß-adrenoceptor activation. Second, the estimation of the RRP in GABAergic nerve terminals using the high-frequency stimulation protocol showed that the ß-agonist increased the RRP both in normal- and high-Ca2+-containing solutions (Fig. 7), suggesting that the increase in the number of releasable synaptic vesicles also contributed to this noradrenergic LTP at BC–PC synapses. The increases in both Ca2+ sensor function and RRP following ß-adrenoceptor stimulation were suppressed by the PKA inhibitors H-89 and Rp-cAMPS (Figs 5 and 6), suggesting that a cAMP–PKA pathway contributes to ß-adrenoceptor-mediated LTP of GABA release. The effect of ISP on the eIPSC amplitude was larger in low Ca2+ while ISP significantly increased the size of RRP even in 5 mM [Ca2+]o. This discrepancy suggests that the Ca2+ sensor was almost saturated in a high-Ca2+ medium, while the replenishment of GABA to releasable pools still occurred during repeated synaptic activation. Alternatively, a high-Ca2+ medium might reduce the excitability at presynaptic nerve terminals, resulting in a less prominent effect of the ß-agonist on the eIPSCs in 5 mM [Ca2+]o than the change in RRP size: the increase in the RRP size could prevent the depletion of releasable GABA associated with massive increases in excitability and release probability following ß-adrenoceptor activation. This interpretation would be consistent with the finding that the ß-agonist also accelerated the recovery from synaptic depression (namely, the refilling of transmitter pools) following high-frequency stimulation (Fig. 7F).
The analysis of GABA release kinetics indicated that ISP facilitated both synchronous and asynchronous GABA release (Fig. 8), which indicated that ISP enhanced the sensitivity of vesicles to both Ca2+ and Sr2+ ions. It is therefore suggested that ß-adrenoceptor stimulation increased the number of synaptic vesicles with both high- and low-release probability and that fast and slow components of GABA exocytosis could be modulated by a shared molecular mechanism underlying the noradrenergic LTP. A similar mechanism has been proposed at chick ciliary ganglion synapses, where noradrenergic protein kinase C (PKC)-dependent mechanism up-regulates the Ca2+ and Sr2+ sensitivities of vesicular proteins, leading to the increase in vesicle fusion probability for synchronous and asynchronous transmitter release (Yawo, 1996). cAMP–PKA cascade has been shown to selectively increase the number of vesicles with higher release probability (Sakaba & Neher, 2001). A synaptic vesicular protein, synaptotagmin, has been shown to regulate only the process associated with fast transmitter release (Geppert et al. 1994). It is therefore unlikely that this vesicular protein could be involved in the ß-adrenoceptor-mediated modulation of asynchronous GABA release.
Possible mechanisms of PKA-dependent enhancement of GABA release
The finding that the PKA inhibitor abolished ß-adrenoceptor-mediated increases in the amplitudes of eIPSCs and HS-induced GABA currents, as well as in the frequency of mIPSCs in PCs, suggested that the noradrenergic LTP at BC–PC synapses was associated with cAMP–PKA-dependent phosphorylation of vesicular proteins that could enhance the translocation of synaptic vesicles towards the active sites in GABAergic presynaptic terminals. Among various vesicular proteins, synapsins, the major substrates of PKA- and Ca2+/calmodulin-dependent protein kinases (Hosaka et al. 1999), have been reported to regulate the synaptic vesicle translocation upon phosphorylation (Hilfiker et al. 1999). Moreover, synapsins have been implicated in modulation of PKA-dependent LTP at hippocampal mossy fibre–CA3 synapses (Rosahl et al. 1995). However, the noradrenergic LTP at cerebellar GABAergic synapses could be induced in synapsin-knockout mice (Saitow et al., unpublished observations). Two other vesicular proteins that serve as PKA substrates are RIM1 and rab3. These two proteins are also unlikely to contribute to the ß-adrenoceptor-mediated LTP of GABA release, because ISP enhanced GABAergic transmission at BC–PC synapses in cerebellar slices derived from knockout mice of these proteins (F. Saitow & S. Konishi, unpublished observations).
Snapin is another PKA target protein of the release machinery members (Ilardi et al. 1999). Although it has been reported in adrenal chromaffin cells that association of synaptotagmin with SNARE complex is enhanced by PKA-dependent phosphorylation of snapin, leading to an increase in both the number of releasable vesicles and the kinetics of the vesicle priming to the release site (Chheda et al. 2001), its roles at central synapses remain unclear. The ß-adrenergic-dependent LTP of GABAergic transmission that we observed at cerebellar BC–PC synapses is likely to result from modulation of vesicular protein interactions via PKA-dependent phosphorylation at the presynaptic active site. Further studies are required to determine the molecular target involved in this form of LTP at inhibitory synapses.
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) and the effect of ß-adrenoceptor antagonist propranolol (•). ISP was applied by superfusion during the period indicated by a black horizontal bar (n = 19). Propranolol (10 µM) was applied during the period indicated by a grey horizontal bar after the ISP-induced enhancement of eIPSCs was fully developed (n = 7). B, the HCN channel blocker ZD7288 abolished a voltage-sag (1) and increased the input resistance (2), resulting in hyperpolarization of a BC (upper trace). Constant current pulses of –200 pA for 400 ms were injected into the BC at 20-s intervals, and ZD7288 was applied by superfusion during the period indicated by a horizontal bar. Records in 1 and 2 were obtained at a and b in the upper trace and displayed on expanded time scales. BCs exhibited repetitive spike firing at the mean resting potential of –51 ± 4 mV (n = 4), which was suppressed by the HCN inhibitor. Note also inhibition by ZD7288 of spontaneous synaptic responses in the BC (see traces in 2). C, inhibition by ZD7288 of spontaneous IPSCs in a PC. Sample records (a and b) represent sIPSCs, obtained at the time points labelled a and b in the upper trace, displayed on an expanded scale. D, the effect of ZD7288 (50 µM) on the ISP-induced enhancement of eIPSCs in PCs. ZD7288 itself caused a partial suppression of eIPSCs (
for 50 Hz, and lines in black). ISP (20 µM) significantly increased the RRP size (• and line in red). D, pooled data of the PPR sizes in the absence (open column) and the presence of ISP (grey column) in 2.5 and 5 mM [Ca2+]o, and the pool size determined with 50-Hz tetanic stimulation. ISP (20 µM) significantly increased the size of the RRP under both [Ca2+]o conditions (2.5 mM [Ca2+]o, P < 0.05, n = 8; 5 mM [Ca2+]o, P < 0.01, n = 8). E, effects of ISP on the rate of transmitter refilling. After conditioning with high frequency stimulation (20 Hz, 30 pulses) in 5 mM [Ca2+]o, single test stimuli with different intervals were applied to evoke IPSCs in a PC and superimposed before (upper trace) and during ISP (20 µM) application (lower trace). ISP accelerated the refilling process. F, pooled data of averaged time course for the recovery of synaptic depression after the tetanic stimulation (20 Hz, 30 pulses) in the control solution (